Penicillin production



Patented Sept. 1951 PENICILLIN PRODUCTION Glenn W. Parsons, Decatur, 11L, asslgnor to A. E. Staley Manufacturing Company, Decatur, 11]., a corporation of Delaware No Drawing. Application June 4, 1949,

Serial No. 97,311 I The present invention relates, generally, to new and useful improvements in the manufacture of penicillin. More specifically, the invention relates to the manufacture of penicillin by culturing penicillin-producing molds in a culture medium containing that part of corn steepwater remaining after removal of solids (largely, salts of phytic acid) precipitated therefrom by mixing therewith under acid conditions a material furnishing aluminum cations in said acidic mixture.

This application is a continuation-in-part of my prior application, Serial No. 633,849, filed December 8, 1945, now Patent No. 2,515,157 issued July 11, 1950.

Phytin is a generic term applied to the salts of phytic acid occurring in plants, and may be obtained as a precipitate when a suitable acidic extract of plant material, such as corn steepwater, is neutralized. The phytin in corn steepwater consists chiefly of a calcium-magnesium phytate.

Salts of phytic acid are also precipitated from corn steepwater when the latter is treated with soluble salts of the alkaline earth metalspor of the heavy metals such as iron and copper. Since phytic acid is polybasic, having a theoretical maximum of 12 available hydrogen atoms, the composition of phytin, with respect to bound metals,'will vary widely, depending upon many different conditions, and especially upon the kind and concentration of metal cations present in the steepwater.

6 Claims. (01. 195-81) for micro-organisms in the production of anti-- biotic substances, being superior to ordinary steepwater with respect to yield of such substances and ease of carrying out the various manufacturing operations. A preferred embodiment of such use is the culturing of Penicillz'um notatum for the production of penicillin. The liquor is also a superior nutrient for the culturing of micro-organisms in general, especially the yeasts, molds, and bacteria. Owing to its lighter color, in comparison with the original steepwater, and to the substantial absence therein of materials that precipitate when it is heated, or neutralized, the liquor is also a superior adjunct in the preparation of foodstufis for human beings and livestock. The precipitate, consisting chiefly of an aluminum magnesium salt of phytic acid, is less contaminated with co-precipitated colored and nitrogenous materials than is the case when phytin is precipitated from steepwater in the usual manner by neutralizing the steepwater with a base. Hence the precipitate is a superior raw material in the preparation of phytic acid and inositol, and for other uses in which the presence of colored and nitrogenous materials would be objectionable.

steepwater is produced when corn starch is manufactured by what is known as the wet-milling process. The first step in this process is to The phytinobtained by neutralizing corn steepwater with a base is contaminated with appreciable proportions of protein-like compounds, in-

organic phosphates, and colored substances. A less contaminated phytin, or salt of phytic acid, is obtained from corn steepwater if it is precipitated from an acidic liquor, i. e., by treating the steepwater at its normal acidity of about 3 to 5 pH with a soluble salt of an alkaline earth metal or of a heavy metal. By this-process the potentially contaminating materials remain dissolved in the acidic mother liquor.

The invention to which my copending application, Serial No. 633,849 is directed, provides a new and useful means for precipitating phytic acid as an insoluble salt in high yield from acidic corn steepwater. That invention embodies the prinsoak the maize kernels in a dilute solution of sulfurous acid at a temperature of from about 110 to 130 F. for a period of time ranging from about 40 to 48 hours. The chief purpose of this soaking or steepin operation is to soften and swell the kernel so as to insure a satisfactory milling operation resulting in a clean separation of starch from germs, bran, and protein. The steeping operation is a fundamental part'of the wetmilling starch manufacturing process-,and the conditions under which this operation is conducted cannot be varied greatlywithout causing trouble throughout the process.

During steeping, the maize kernels gradually swell and soluble materials therein pass'into the steepwater. The sulfurous acid present serves a dual purpose; it inhibits the growth of putrefactive organisms in the steepwater, and it exerts a speicfic loosening effect upon the proteinous material that binds the starch granules together in ciple of dividing the steepwater into two parts, a

liquor and a precipitate, both of which have superior qualities with respect to many different uses. It was found, according to the present invention, that the liquor is an excellent nutrient 3 of the dissolved substances in the steepwater, contributes to the softening effect on the kernels and is a distinct acid to the subsequent milling operations.

Corn may be steeped batchwise, or in a countercurrent system, or by a combination of the two methods. The sulfurous acid concentration in the initial steeping water is made up to the desired.

value of about 0.2 per cent (calculated a sulfur dioxide) by passing sulfur dioxide gas into makeup water, after which no more is added. The initial pH of the steepwater is low (below 4) because of its sulfurous acid content. Much of the sulfurous acid disappears after a few hours steeping. Meanwhile lactic acid fermentation begins and generation of this acid keeps the steepwater pH down around a value of about 4. As the solubles content of the steepwater increases, the steepwater density rises until at the end of the operation it lies within the range of about 3 to 7 Baum, the final value depending to a considerable extent upon the steeping system used. The steepwater thus produced, known as light steepwater, is sent to evaporators and concentrated to "heavy" steepwater having a density of about 25 to 30 Baum. The bulk of the heavy" steepwater has been mixed, in the past, with the corn gluten and bran as an important ingredient in corn gluten stock feed. Occasionally some heavy" steepwater has been sold to yeast growers as a nutrient for the yeast.

Corn steepwater contains an extremely complex mixture of substances among which are protein hydrolytic products of assorted molecular weights, reducing sugars, lactic acid, phytln, B complex vitamins, organic acids, amines, and inorganic anions and cations. Undoubtedly the steepwater also contains many other substances that have not yet been detected therein. Concentrated steepwater of 30 Baum density contains about 52 per cent dry substance by weight. It is a brown, viscous liquid which upon standing at room temperature deposits a soft sludge consisting in part of protein-like material, magnesium and calcium lactates and calcium and magnesium phy tates.

In regard to the use of corn steepwater as a nutrient for molds of the genus Penicillia, in the manufacture of penicillin, it appears that the steepwater contains one or more substances which exert a specific effect upon the mold. That is, the mold is caused to produce mroe penicillin than it otherwise would during normal growth in the usual synthetic media containing salts, carbohydrates, and nitrogen compounds. As an alternative possibility, the steepwater may contain an unusually favorable combination of growth factors in its vitamins, amino acids, carbohydrates, and salts that is responsible for the increased penicillin production by the mold.

Penicillin is produced by preparing a culture broth for a selected penicillium mold, commonly Penicillium notatum, comprising a dilute aqueous solution of nutrient ingredients including salts, sugars, and nitrogen compounds, sterilizing the culture medium by autoclaving it, cooling the medium, inoculating it with spores of the mold, allowing the mold to grow under optimum conditions of aeration, pH, and temperature until the penicillin content of the culture medium reaches a maximum value, and recovering penicillin from the medium by suitable extraction and concentration procedures.

The outstanding desirable, but it has several drawbacks that have caused trouble. For one thing, steepwater contains much extraneous material that is either of no benefit or injurious to the penicillin-producin: ability of the mold, and interferes with the recovery of penicillin from the broth culture. Also, as mentioned before, the concentrated steepwater deposits a sludge which at times becomes almost solid. upon standing. This behavior has been a continuous source of difficulty in transporting and storing the material. It is not feasible to provide a steepwater of lighter density, containing more water, because the less concentrated liquor is extremely susceptible to a bac- ,material coats surfaces such as the insides of glass bottles used in the surface culture method of manufacturing penicillin, and is diflicult to scrub off afterward. In addition, as the mold grows, the pH of the culture medium rises to values around '7 to 8 with the result that calcium and magnesium salts of phytic and phosphoric acids, and other materials, are precipitated which interfere in the subsequent recovery of penicillin from the medium.

Precipitation of phytln from corn steepwater by moderately raising the pH with caustic soda eliminates the foregoing disadvantages of the steepwater as a mold nutrient. Such precipitation. however, reduces the nutrient activity of the liquor in an amount that is proportional to the increase in liquor pH. This indicates that possibly some steepwater ingredient, essential to high production of penicillin by the mold, is being progressively removed with the phytln precipitate as the liquor pH is raised. It was conceived that in order to correct the aforementioned objectionable features of untreated corn steepwater without impairing its nutrient activity, adequate amounts of phytln should be removed from it at low pH. possibly at its normal acidity of about 3 to 5 pH. A search for satisfactory methods to accomplish such removal of phytln was not successful until it was unexpectedly discovered that aluminum cation was an excellent precipitant of phytln from the acidic steepwater.

The present invention eliminates all the foregoing disadvantages of corn steepwater as a mold nutrient in the production of penicillin and provides for an increase in the yield and quality of penicillin over that obtained with ordinary corn steepwater.

Among the objects of this invention are the following, all relating to treatment of corn steepwater:

1. Provision of improved culture media for the culturing of microorganisms in the production of antibiotic substances.

2. Provision of superior culture media for the growth of penicillin-producing molds.

3. Provision of improved culture media for utility of corn steepwater in penicillin-producing molds which do not yield an the production of penicillin has made its use most 15 o iectio able precipitate when the media is sterilized by autoclavingpriol to its inoculation with spores of themolds. j v

4. Provision of culture media for penicillinproducing molds which do. not yield an objectionable precipitate in themedia during growth of the molds.

5. Provision of clean culture media for penicillin-producing molds which result in higher recovered yields and quality of penicillin because of the freedom from troublesome impurities.

6. An improvement in the process of producing penicillin by fermenthtion which comprises incorporating in the culture media an aqueous nutrient material obtained by treating corn steepwater with a material furnishing aluminum cations, and separating the supernatant liquor from the resulting precipitate.

Other objects of the invention will in part be obvious and will in part appear hereinafter.

The invention accordinglycomprises the several steps and the relation of one or more .of such steps with respect to each of the others, and the product possessing the features, properties, and the relation of constituents, which are exemplified in the following. detailed disclosure, and the scope of the application. of which will be indicated in the claims.

Briefly, the inventionto which my copending application, Serial No. 633,849, is directed, contemplates mixing corn steepwater at its normal acidity of about 4 pH with a material which will furnish aluminum cations in the acidic liquor, heating the mixture. to promote the most efilcient precipitation of the aluminum-magnesium phytate, and separating liquor from precipitate by any suitable means,

The treatment of the steepwater may be carried out as follows: Corn steepwater at about 4 pH and concentrated to about. Baum is placed in a vessel equipped with an agitator and a heating device. Ordinary granular alum (hydrated aluminum sulfate) in amount up to about per cent of the weight of steepwater solids, is stirred into the steepwater. The addition of the alum produces an immediate precipitate of aluminummagnesium phytate. The mixture is stirred and heated to about 70 C. to promote the phytin precipitation. It is then immediately filtered while hot. The filter cake is washed free of mother liquor with warm water, and the washings are combined with the main filtrate.

The wet filter cake may be ,llsed as is, or may be dried beforehand. Likewise, the .liquor may be used directly in its diluted state, but in general,'it will prove more desirable to concentrate it further to inhibit spoilage and to reduce storage and shipping costs. Preferably the concen-,

tration is conducted under reduced pressure and carried to a liquor density of about 30 Baum. Additional solids, mostly lactic acid salts, separate during and after the concentration. For some uses, it may be desirable to remove .these from' the liquor. This-is preferably accomplished by cooling the concentrateto about C., agitating it gently for a clay or so, and then filtering or centrifugingit. The clarified liquor is a stable liquid product that has a greatly reduced tendency to mold or spoil during storage. It is nontoxic, free of materials that precipitate when it is heated, lighter in color than untreated steepwater concentrated to the same degree, or equally concentrated steepwater from which the phytin has been precipitated with alkalineagents such as lime or caustic soda.

Permissible adjustment'of'variable factors in the process, within ranges that provide a satis- I 6 factory'treatment of the steepwater, will appear in the following discussion of the individual variables.

Raw materiaL-The corn steepwater used mayvary considerably in its history as to:

(a) Fermentation.-It may have been fermented with Lactobacilli to widely varying degrees; i. e., conversion of soluble sugars to lactic acid may vary from 0 to 100 per cent. Regarding use of the liquid product of corn steepwater treated according to this invention in the' pro-- duction of penicillin, however, it has been found that highly fermented steepwater provides higher yields of penicillin by the submerged growth method than does the unfermented or partially fermentedkind. For example, with only about 2 per cent. reducing sugars in the Baum liquid product, by virtue of prior fermentation of the sugars with Lactobacilli commonl present in corn steepwater, the yield of penicillin is about 20 per cent higher than it is when the 30 Baum liquid product contains about 8 per cent reducing sugars.

I (b) Heat treatment-The steepwater may have been heated to widely varying temperatures, for example, up to C. in concentrating it or removing volatile constituents, such as sulfur dioxide.

(c) steeping method.-It may have been obtained by any of the commercial'corn steeping processes, whether of the continuous or batch type.

mixture of aluminum compounds is satisfactory, provided itv furnishes a concentration of aluminum cations suillcient to produce a precipitate; and provided further thatit does not introduce objectionable anions, such as those that are toxic to microorganisms and to animals. Because of their cheapness an non-toxic anions, ready availability. and ease in handling, aluminum sulfate .and aluminum chloride are the preferred aluminum compounds. All that is required of the aluminum compound used is that it react with the steepwater components in a suitable fashion, by yielding a concentration of aluminum cations great enough to exceed the solubility product of the aluminum-magnesium phytate and thus cause its precipitation. Thus, hydrated aluminum oxide, or so-called aluminum hydroxide, despite its very low solubility in water alone, operates satisfactorily in the normal acid medium of steepwater because it is suiliciently soluble therein to provide the required concentration of aluminum cations. Also, since aluminum metal yields aluminum cations in acid media, aluminum metal itself may be used as a precipitant for 1.6 per cent aluminum. Suitable proportions of other aluminum compounds, such as the nitrate, chloride, and hydrated oxide, when used, lie, for

example, in the range of 0.08 to 1.6 per centv aluminum based on the steepwater dry substance. In general, any proportion of aluminum cation that produces a precipitate may be used. The proportion selected will vary with the intended use of the products.

Precipitant.-Any aluminum compound or The influence of proportion of precipitant on yield of precipitate is indicated by the following dry weights of washed precipitate obtained by adding varying amounts of hydrated alum to 100 ml. of 30 Baum steepwater at 4 pH and heated to 100 C.; 2.4 gms. at 2.5 per cent alum (based on steepwater solids); 5.1 gms. at 5.0 per cent alum; 7.5 gms. at 7.5 per cent alum; 11.1 gms. at 10.0 per cent alum; 17.3 gms. at per cent alum; and 20.4 gms. at per cent alum.

When a high yield of the aluminum-magnesium phytate is the primary interest, it will be desirable to use a high proportion of alum, but if uses of the remaining liquid portion of the steepwater are of primary interest, it may be desirable to employ low proportions of alum. This is especially true if nutrient uses of the liquor are considered. For example, with respect to use of the liquor as a nutrient for penicillium mold in the production of penicillin in accordance with the present invention, it is not desirable to remove all the phytin from the steepwater. To do so reduces its value as a mold nutrient. Instead, it is desirable that only enough phytin be removed to overcome the earlier stated difiiculties associated with the use of ordinary corn steepwater as a nutrient for penicillium mold.

Temperature.-Temperature at which the steepwater is treated with the aluminum compound may vary over a wide range, for example, from 0 to 100 C. The preferred range is from about 50 to 80 C. Below 50 C. the effect of temperature on precipitation of the aluminummagnesium phytate is not large, and although precipitation of the salt does increase with increasing temperature above 80 C., darkening of the liquor sets in at around 80 C. and becomes quite noticeable at 100 C.

The following dry weights of washed precipitate obtained by adding 8 per cent hydrated alum (based on the steepwater dry substance) to 100 ml. of Baum steepwater at 4.3 pH, and at varying temperatures, illustrate th effect of temperature on the precipitation: 3.6 gms. at 20 C.; 4.0 gms. at C.; 6.1 gms. at 60 C.; 6.8 gms. at 80 C.; and 7.2 gms. at 100 C. 0

pH of treatment.--As pointed out earlier. precipitates of phytic acid salts obtained from acidic corn steepwater are less contaminated with objectionable impurities than is the case when the precipitation occurs under neutral or alkaline conditions. Optimum results are obtained in the pH range ofabout 3 to 5, but satisfactory results. are secured with acidic steepwaters in general; i. e., at pH values from about 0.5 to 7.

. Because of its slight solubility in acid, the yield of aluminum-magnesium phytate precipitated from corn steepwater according to this invention decreases slowly with decreasing pH of the mother liquor. For example, the following dry weights of washed precipitate were obtained by adding 8 per cent hydrated alum (based on steepwater dry substance) to 100 ml. of 30 Baum steepwater at 100 C. and adjusted to varying pH values with hydrochloric acid and sodium hydroxide: 6.2 gms. at 3.1 pH; 6.8 gms. at 3.6 pH; 7.3 gms. at 4.0'pH; and 7.6 gms. at 4.5 pH.'

The pH of corn steepwater normally'lies within the range of about 3 to 5. and is usually in the neighborhood of about 4. Addition of an acidreacting aluminum salt, such as aluminum sulfate or aluminum chloride, to corn steepwater at 4 pH lowers its pH only a few tenths of a unit.

Density of treated steepwater.'This may varyfrom that of the original light steepwater, about 3 Baum up to the final desired value of about 30 Baum, but the preferred value is around 15 Baum. v

Duration of treatment.-Precipitation of the phytate is rapid, especially at elevated temperature, and the treated steepwater can be filtered as soon as it has been heated to the desired tem perature. There is no disadvantage to delay in filtration, however, unless the mixture be held hot for extended periods of time. In this case, slight darkening of the liquor may occur.

The following specific example will illustrate the treatment: 2,000 gallons of 15 Baum steepwater, obtained by vacuum evaporation of a larger volume of 4 Baum steepwater, were placed in a tank equipped with an agitator and a steam coil. To this liquor, having a pH of'4.0 and containing 4,440 pounds of dry substance. was added 222 pounds of granular hydrated aluminum sulfate (papermakers alum) with stirring and heating to 72 C. Precipitation of the phytate' ceased when all the alum had been added and the mixture had been stirred for a few minutes at 72 C. The mixture, now at apH of about 3.8, was filtered hot in a filter press. The filter cake was washed with warm water in the press until it was substantially free of mother liquor, and the washings were combined with the main filtrate. The weight of wet washed filter cake was 1,200 pounds (500 pounds of dry substance), the volume of main filtrate was 1,880 gallons, the washings amounted to 370 gallons, and the volume of combined main filtrate and washings was 2,250 gallons.

For some purposes, as in the preparation of phytic acid and of inositol, the filter cake obtained in the preceding example may be used in its wet state. For other purposes, it may be desirable to dry the cake beforehand.

The filtrate, or the combined filtrate and washings, obtained in the preceding example may also be used as it is for manypurposes. According to the present invention it is used as a superior nutrient for the growth of penicillium mold in the production of penicillin. In case the liquor is not used immediately, it will generally be desirable to concentrate it to a density of about 30 Baum. As mentioned above, this will greatly reduce danger of its spoilage by putrefaction or undesirable fermentation, and will reduce storage and shipping costs. Preferably the concen-,

tration should be carried out under reduced pressure. Crystalline solids, mostly salts of lactic, acid, will appear in the liquor during and after, its concentration. Their removal is optional, but. usually desirable, because sometimes their pres-- ence causes the concentrated liquor to become semi-solid and difiicult to pump or transfer.

The proportions of bound aluminum and magnesium in the phytic acid salt obtained by the process of this invention will vary, depending upon many factors. especially the quality of the steepwater and the proportion of added aluminum compound.

All liquor densities mentioned hereinbefore andi then neutralizing it with caustic soda solution.

liqnlto P 9. v is directed. 'andconsisting of that principle of using phenolphthalein as indicator, and (stall fuglng off the precipitated zinc hydroxide.

"It has .been "found that the com steepwater nutrient, pr pared in accordance with" the invenapplication, Serial No. 633,849,

' "to" raise the yields of penicillin when manufactured according to the several known fermenta= tion techniques. 1 Thus,incorporation of this improved corn steepwater nutrient in the culture media is beneficial, whenthe fermentation'is of the surface type or of the submerged (i. e. deep tank) type. Any one of the several known penicillin producing molds may be used, including, Penicillium notatum, P. chrusoyenum, P. cyaneojulvum, and P. baculatum. 'The specific compositions of the culture media may vary with respect to the particular individual nutrient ingredients present and the concentrations thereof. Referring to the following examples, the utility of my invention may be shown by producing penicillin according to several different typical techniques using typical culture media, and carrying out duplicate fermentations in each instance except that in one set of fermentations a typical corn steepwater nutrient is used while in the duplicate set of fermentations this same corn steepwater is used after first having been treated in accordance with the invention to which my application, Serial No. 633,849, is directed.

Example 1 A culture medium is formulated having the following composition:

Lactose gm 37.5 MgSO4.7I-I2O gm .25 NaNOa m 3.0 Commercial glucose gm 2.75 ZnSOdIHzO gm .05 30 B. corn steepwater -l ml 75 H20 q. s liter 1 (pH is adjusted to 4.4 with KOH) 1249, and then fermented under the same condi-.

tions of still surface fermentation for 8 days. The. yields in Oxford units obtained runnin three sets of surface fermentations are as follows.

Untreated Treated Per cent g gg g g Com Steepincrease in 01) water yield following composition:

10 Example 2 a, culture medium; is formulated hayin the same composition.isprepared,replacing the untreated corn steepwater; with 5 an equal quantity from the same source which has been. processed with aluminum sulfa-te according 1 to the procedure described above.

Each liter of the culture media is placed in a tank type fermenter provided with an efficient agitator and an air distributor. After being sterilized in situ, the culture media are cooled and inoculated with equal spore concentrations of P. chrysogenum X-1612. The media are fermented for 3 days during which aeration and agitation are continuously maintained.

A series of test fermentations of this type show that the yields of penicillin are increased from 15 to 150 per cent when my specially treated corn steepwater nutrient is used in place of the untreated corn steepwater nutrient.

Since certain changes in carrying out the above process, and certain modifications in the product which embody the invention may be made without departing from its scope, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense.

It is also to be understood that the followin claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween.

. Having described my invention, what I claim as new and desire-.to secure by Letters Patent, is:

'1. In the manufacture of penicillin by the culturing of penicillin-producing molds, the improvement which comprises culturing a penicillin-producing mold in a culture medium contaming as a nutrient that principle of corn steepwater remaining after removal therefrom of phytate solids precipitated by mixing therewith under acid conditions a material furnishing aluminum cations in said acidic mixture.

2. In the manufacture of pencillin by the cuituring of penicillin-producing molds, the improvement which comprises culturing a penicillin-producing mold in a culture medium containing as a. nutrient that principle of fermented corn steepwater remaining after removal of phytate turing of penicillin-producing molds, the improvement which comprises culturing a penicillin-producing mold in a culture medium containing as a nutrient that liquid product obtained from corn steepwater by mixing with the steep water under acid conditions a material furnishing aluminum cations therein in amount sumcient ll to cause precipitation or an aluminum-containing salt of phytic acid irom the mixture when it is heated, heating said mixture to a temperature of not less than about 50C. to cause a precipitation or said salt, separating liquor from precipitate and rejecting said precipitate, concentrating said liquor to a density of not less than about 30 Baum, subjecting said liquor to a crystallizing step whereby solids separate from the liquor, separating said solids from liquor, and rejecting said solids.

4. The improvement as defined in claim 1 in which the source of aluminum cations is aluminum sulfate. 3

5. The improvement as defined in claim 1 in Number num nitrate.

GLENN W. PARSONS.

I REFERENCES CITED The following references are of record in the 10 file of this patent:

UNITED STATES PATENTS Name Date 2,467,107 Moyer July 12, 1949 

1. IN THE MANUFACTURE OF PENICILLIN BY THE CULTURING OF PENICILLIN-PRODUCING MOLDS, THE IM-PROVEMENT WHICH COMPRISES CULTURING A PENICILLIN-PRODUCING MOLD IN A CULTURE MEDIUM CONTAINING AS A NUTRIENT THAT PRINCIPLE OF CORN STEEPWATER REMAINING AFTER REMOVAL THEREFROM OF PHYTATE SOLIDS PRECIPITATED BY MIXING THEREWITH UNDER ACID CONDITIONS A MATERIAL FURNISHING ALUMINUM CATIONS IN SAID ACIDIC MIXTURE. 